The CD8 cell surface glycoprotein is expressed on subsets of T cells, natural killer cells, and dendritic cells at various stages of differentiation. It functions as both an adhesion and a signaling molecule on cytotoxic and regulatory T cells. CD8 is expressed as either an alpha/alpha (a/a) homodimer or as an alpha/beta (a/b) heterodimer. These two forms of CD8 are differentially expressed on certain cell types and are functionally distinct. One of the CD8 ligands is the major histocompatibility class I molecules to which the T cell receptor also binds. The goals of the proposal are to determine the structural basis for CD8-MHC class I interaction in molecular detail and to determine if there are differences in this interaction between the two forms of CD8 that may account for their functional differences. Another goal is to determine the structural basis for the interaction between CD8 on regulatory T cells with a 180 kD ligand expressed on intestinal epithelial cells. Differences in interaction between MHC class I and CD8 on cytotoxic cells vs. the 180 kD protein and CD8 on regulatory T cells may be partially responsible for alternative pathways of T-cell activation and thus functional differences between these two cell types. A new class of antigen cell surface molecules which present antigens to T cells is the CD1 family of proteins. These proteins appear to be ligands for CD8. It is planned to determine the structural basis for these interactions as well. A combination of approaches will be used for these studies. Cell-cell adhesion assays between cells transfected with either CD8 or ligands will be performed to demonstrate direct interactions. Mutagenesis studies will be used to identify critical residues for protein-protein interactions and complementary mutational analysis for determining topology of the interactions. Affinities and association and dissociation rate constants will be determined using surface plasmon resonance detection with a BIAcore. The work proposed will extend understanding of the basic mechanism of immune recognition, a crucial part of host defense against cancer and infection. Clinically, the structure/function studies carried out in this proposal will potentially allow for the design of therapeutic proteins bearing CD8 Ig-like domains that may be used to modulate specific CD8-ligand interactions and the T-cell activation events which are triggered by these interactions with CD8.